Chemotactic peptide antagonists for imaging sites of inflammation

ABSTRACT

Radiopharmaceuticals comprising molecules that target to N-formyl-methionyl-leucyl-phenylalanine (fMLF) receptor on leukocytes in order to target sites of inflammation for diagnostic imaging are described. The targeting molecules are attached to capping groups that make the entire molecule either antagonists or weak agonists of fMLF receptor and therefore do not elicit a chemotactic response resulting in neutropenia. The preferred targeting molecule is ReO-Gly-lys(Dimethylgly-t-Butylgly-cys-gly)-glu-trp-phe-leu-nle-NHCOcyclopropyl. The invention also relates to the use of combinatorial chemistry to obtain preferred molecules that target sites of inflammation for diagnostic imaging.

FIELD OF THE INVENTION

The present invention relates to diagnostic imaging of the sites ofinflammation in humans and animals. More particularly, the presentinvention relates to the use of combinatorial chemistry to identifytargeting molecules which bind to receptors on cells that accumulate atsites of inflammation.

BACKGROUND OF THE INVENTION

The art of diagnostic imaging exploits contrasting agents that inbinding or localizing a site selectively within the body, help toresolve the image of diagnostic interest. ⁶⁷Gallium salts, for example,have an affinity for tumours and infected tissue. With the aid ofscanning tomography, ⁶⁷Gallium salts can reveal afflicted body regionsto the physician. Other contrasting agents include metal radionuclidessuch as ^(99m)technetium and ^(186/188)rhenium. These have been used tolabel targeting molecules such as proteins, peptides and antibodies thatlocalize at desired regions of the human body.

It is critical for the management of patient care to have the ability toquickly and accurately identify sites of infection and resultantinflammation. Current radionuclide labeled targeting molecules fortargeting sites of infection and resultant inflammation bind toleukocytes (white blood cells). These targeting molecules do not provideprompt diagnosis due to delays of up to 12-24 hours following injection.These delays occur because the targeting molecules require theseparation of leukocytes from the patients whole blood beforeradiolabeling. Further delays result because after re-injection theleukocytes take several hours before they can re-localize to the sitesof inflammation. Other targeting molecules have high molecular weightwhich results in slow delivery of the radiopharmaceutical to the sitesof inflammation.

The problem with time delays can be overcome by usingradiopharmaceuticals that bind well toN-formyl-methionyl-leucyl-phenylalanine (fMLF) receptor on leukocytes.This receptor is also referred to as formyl peptide receptor (FPR). Theaccumulation of leukocytes at sites of inflammation is the primarymechanism by which the immune system localizes and destroys microbialand other toxic agents. Radiopharmaceuticals that bind to fMLF receptorcan label leukocytes that are present in circulation as well as those atsites of inflammation. This allows for prompt diagnosis.

fMLF is a bacterial product that initiates leukocyte chemotaxis bybinding to high affinity fMLF receptors present on polymorphonuclearleukocytes (PMNs) and mononuclear phagocytes. Chemotaxis refers to themigration of leukocytes to sites of inflammation and infection. Currenttargeting molecules that that bind well to fMLF receptor are agonistsfor this receptor. These targeting molecules therefore elicit anaccumulation of leukocytes in healthy tissue that causes tissue damage.This effect is called neutropenia. This is a significant problem.

Most known chemotactic antagonists however exhibit low binding affinityto the fMLF receptor. None have a group that is suitable fortransporting a radionuclide that is essential for radioimagingapplications.

There is therefore a need for radiopharmaceuticals that have a highaffinity for the fMLF receptor so that they can target leukocytes thataccumulate at sites of inflammation. There is a further need for suchradiopharmaceuticals that are antagonists or very weak agonists ofchemotaxis in order to eliminate problems such as neutropenia that areassociated with agonists.

Recent studies have indicated that the binding affinity of targetingpeptides and the chemotactic functionality of those targeting peptidesdepend on two different factors. The three amino acid residues at theN-terminus of the peptides (methionine, leucine and phenylalanine)mainly govern the degree of binding of the targeting peptide to thereceptor. There is also a contribution from amino acid residues that aremore removed from this site. The functional group attached to theN-terminus of the peptides has a large affect on the chemotacticfunctionality of the peptide when bound to the receptor. For example thepresence of a formyl group or an acetyl group has been shown to provideagonist or weak agonist activity whereas a tertbutoxycarbonyl groupcauses the peptide to exhibit antagonist activity but with low affinitybinding.

There is therefore a need for a radiopharmaceutical that is achemotactic antagonist with an N-terminus capping group and a structurethat confers a strong binding affinity for the fMLF receptor to theradiopharmaceutical.

SUMMARY OF THE INVENTION

The invention relates to radiopharmaceuticals that are capable ofcomplexing a radionuclide metal and that has a high binding affinity forfMLF receptor. The radiopharmaceuticals of the present invention areantagonists or weak agonists of chemotaxis.

The invention includes metal-containing molecules that are capable ofbinding to the fMLF receptor. The invention also includes molecules thatcontain a region complexed to transition metals. The presence of themetal within the molecule allows for the accumulation of the metal atsites of inflammation or infection, since the whole molecule binds toreceptors upregulated on leukocytes that accumulate at these sites, andthus allows for the detection of inflammation or infection.

According to one aspect of the present invention, there is provided acombinatorial library for obtaining compounds that target sites ofinflammation comprising a mixture molecules of the following formula I:

wherein CG is a capping group that makes the compound an antagonist or aweak agonist to chemotaxis; X, X2, X3 and X4 are amino acids selectedfrom natural and unnatural amino acids; Z is a chelator capable ofcomplexing a radionuclide metal or a chelator attached to a radionuclidemetal, X3 being a site of attachment for said chelator.

According to another aspect of the present invention, there is provideda compound for binding to sites of sites of inflammation having thefollowing formula I:

wherein CG is a capping group that makes the compound an antagonist or aweak agonist to chemotaxis; X, X2, X3 and X4 are amino acids selectedfrom natural and unnatural amino acids; Z is a chelator capable ofcomplexing a radionuclide metal or a chelator attached to a radionuclidemetal, X3 being a site of attachment for said chelator.

According to a particular aspect of the invention there are providedcompounds of formula II:

wherein CG is a capping group that makes the compound an antagonist or aweak agonist to chemotaxis; X, X2, X3 and X4 are amino acids selectedfrom natural and unnatural amino acids; X3 being a site of attachmentfor said chelator, R, and R2 is a linear or branched, saturated orunsaturated C₁₋₆ alkyl chain that is optionally interrupted by one ortwo heteroatoms selected from N, O, and S; and is optionally substitutedby at least one group selected from hydroxyl, amino, carboxyl, C₁₋₆alkyl, aryl and C(O)R; R3 is selected from H; alkyl; an alkylsubstituted by a group selected from amino, aminoacyl, carboxyl,guaniginyl, hydroxyl, thiol, phenyl, phenolyl, indolyl, and imidazolyl;

According to another aspect of the invention there are providedcompounds of the formula III:

wherein CG is a capping group that makes the compound an antagonist or aweak agonist to chemotaxis; X, X2, X3 and X4 are amino acids selectedfrom natural and unnatural amino acids; X3 being a site of attachmentfor said chelator; and R, R2 and R3 are defined as above; M is a metalor an oxide or nitride thereof.

According to yet another aspect of the present invention, there isprovided a method of obtaining a compound that targets sites of sites ofinflammation comprising the steps of:

-   I) Preparing a library of compounds of the following formula I:    wherein CG is a capping group that makes the compound an antagonist    or a weak agonist to chemotaxis; X, X2, X3 and X4 are amino acids    selected from natural and unnatural amino acids; Z is a chelator    capable of complexing a radionuclide metal, X3 being a site of    attachment for said chelator.-   II) Preparing mixtures of said compounds comprising different    variables of said natural or synthetic amino acids and said capping    groups;-   III) Testing said mixtures for binding to    N-formyl-methionyl-leucyl-phenylalanine (fMLF) receptor,-   IV) Selecting mixtures that show positive binding to    N-formyl-methionyl-leucyl-phenylalanine (fMLF) receptor, and-   V) Deconvoluting said mixtures that show positive binding to    N-formyl-methionyl-leucyl-phenylalanine (fMLF) receptor to obtain a    compound that binds to N-formyl-methionyl-leucyl-phenylalanine    (fMLF) receptor.

According to another aspect of the invention there is provided a use ofthe compound of formula I to target and image sites of inflammation in apatient.

The invention also includes a kit comprising compounds of formula I, andsuitable reagents for labeling the compounds with a radionuclide metaland delivering the labeled compounds to a patient.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph showing competition Binding of Re-library mixturesadministered to elicited rat PMNs in the presence of 6 nM H³-fMLF. Datais expressed as a % control of ³H-fMLF binding with standard errorsbeing ≦15% in all cases (n=1; replicates of 4). FIG. 1A representsmixtures administered at 10 uM concentrations. The control compoundillustrates competition binding by ReORP455 (Re-metal complexed to the(Dimethylgly-t-Butylgly-cys-gly)-chelator) while FIG. 1B representscompetition binding of library mixtures administered at 1.0 uMconcentrations;

FIG. 2 shows competition-binding curves of library ReORP553deconvolution compounds with 6 nM ³H-fMLF. Fit of curves to one-sitecompetition function with Prism software is indicated for a correlationcoefficient ≧0.98 for all graphs. ReORP553 17-01 exhibited the greatestbinding affinity and the receptor binding curve in relation to controlcompounds fMLF and N-t-BocMLF which is represented in graph A;

FIG. 3 is a binding curve showing competition binding of ReORP553 17-01with 6 nM ³H-fMLF to human neutrophils. Fit of curves to one-sitecompetition function with Prism software indicated a correlationcoefficient ≧0.97 for all binding curves. ReORP553 17-01 exhibited abinding affinity which is similar to N-t-BocMLF and less than fMLF (n=1,performed in replicates of 3);

FIG. 4 is a bar graph showing the effect of ReORP553 17-01 on MPOrelease in human neutrophils stimulated with varying concentrations offMLF. Significant inhibition of MPO release is observed by 10 uMReORP553 17-01 at fMLF concentrations of 10⁻⁸ M and 10⁻⁶M. Data isexpressed as percent of control absorbance ±S.E.M. (n=1, performed inreplicates of 4) This effect is comparable to that of N-t-BocMLF, aknown antagonist

FIG. 5 is a plot showing HPLC purified Tc-99m-RP553 17-01 labeling.RP553 17-01 was radiolabeled with 99m sodium pertechnetate under RP55317-01 (200 ug, 0.144 mmoles) was dissolved in 100 μl saline and 100 μlacetonitrile. Tin (II) chloride (40 ug) and sodium gluconate (1.3 mg)were added to the peptide solution followed by Na[⁹⁹TcO₄] (10 mCi). Thesolution was carried out for 1 hour at room temperature. The reactionwas analyzed using HPLC and the product was purified under astep-gradient condition. The radiochemical purity of the HPLC purifiedTc-99m-RP553 17-01 was >90% as shown in the trace;

FIG. 6 is a plot showing binding of f-Met-Leu-[3H]Phe to elicited ratperitoneal neutrophils. Neutrophils (0.25×10⁶-1.0×10⁶ cells) wereincubated at 4° C. for 1 hour with varying concentrations off-Met-Leu-[³H]Phe. For FIG. 6A the data represents specific binding(total minus nonspecific binding in the presence of excessnonradiolabeled fMLF) (r=0.95). For FIG. 6B the data representsScatchard transformation of the binding isotherm indicates binding to asingle receptor population with a slope of −0.16±0.02 and an x-interceptof approximately 34±0.5 (r=−0.97; n=3 performed in quadruplicate);

FIG. 7 is a plot that shows the release of MPO by elicited ratperitoneal neutrophils. Dose-dependent release of myeloperoxidase wasexhibited by fMLF in FIG. 7A and fMLFK in FIG. 7B. Agonist response isexpressed as % of the O.D.₄₉₀ value attained in control wells lackingstimulation and error bars represent S.E.M. values (n=3, performed inquadruplicate; r=0.99); and

FIG. 8 is a bar graph showing competition Binding of N-terminal modifiedpeptide NleLFK to elicited rat PMNs. Modified peptides were administeredat 1 uM and 10 uM concentrations in the presence of 6 nM ³H-fMLF. Theiso refers to administration of known antagonist iso-BocMLFK. Data isexpressed as mean % control with standard errors being <15% in all cases(n=1, performed in quadruplicate).

DETAILED DESCRIPTION

The radiopharmaceuticals of the present invention are molecules thattarget sites of inflammation. The preferred molecules are peptides thatbind fMLF receptor. The molecules include a chelating moiety thatcomplexes radionuclide metals such as ^(99m)Tc and Re which isisostructural to ^(99m)Tc. The chelating moiety may play a role inbinding fMLF receptor.

The molecules of the of the present invention have the followingformula:

wherein CG is a capping group that makes the compound an antagonist or aweak agonist to chemotaxis; X, X2, X3 and X4 are amino acids selectedfrom natural and unnatural amino acids; Z is a chelator capable ofcomplexing a radionuclide metal or a chelator attached to a radionuclidemetal, X3 being a site of attachment for said chelator.

X3 is the amino acid residue used to attach the metal preferably hasthree functional groups. Preferably X3 is lysine. Amino acid X3 musthave a point of attachment to the growing peptide to facilitatesynthesis. Such a moiety allows a suitably protected reagent to reactwith the amino terminus of the solid supported peptide during thesynthesis (in the case of lysine this is the carboxyl group). Theresidue must also contain a group for the attachment of further aminoacids to enable the peptide synthesis to be continued. In the case ofthe preferred lysine this is the □-amino group of the amino acid.Finally, the reagent must incorporate a functional group for the laterattachment of the metal chelating portion of the molecule. This may beany group capable of attaching to a metal chelator and may also belinked to the rest of the residue by aliphatic, aromatic,heteroaromatic, ether or other similar groups. These groups may or maynot be involved in the binding of the molecule to the fMLF receptor. Inthe case of lysine therefore the three groups are carboxyl, amino andamino. In some cases (for example lysine) it may be necessary tooptionally protect one or two of the groups to allow selective reactionsto take place. Subsequent deprotection will then furnish the requiredfunctional group for further reaction.

The preferred amino acid for X4 is glycine. Other amino acids may alsobe used at position X4.

The metal chelating moiety used for the purposes of the presentinvention includes chelators having the following general formula:

-   -   wherein,    -   X is a linear or branched, saturated or unsaturated C₁₋₆ alkyl        chain that is optionally interrupted by one or two heteroatoms        selected from N, O, and S; and is optionally substituted by at        least one group selected from hydroxyl, amino, carboxyl, C₁₋₆        alkyl, aryl and C(O)R;    -   Y is H or a substituent defined by X;    -   Z is the position of attachment for the targeting portion of the        library; R¹ through R⁴ are selected independently from H;        carboxyl; C₁₋₄ alkyl; C₁₋₄ alkyl substituted with a group        selected from hydroxyl, amino, sulfhydryl, halogen, carboxyl,        C₁₋₄ alkoxycarbonyl and aminocarbonyl; an alpha carbon side        chain of a D or L-amino acid other than proline; and C(O)R;    -   R⁵ is selected from H and a sulfur protecting group; and    -   T is carbonyl or CH₂.

Where the chelator is complexed to a metal or a metal radionuclide, thecomplex has the following general formula:

-   -   X is a linear or branched, saturated or unsaturated C₁₋₆ alkyl        chain that is optionally interrupted by one or two heteroatoms        selected from N, O, and S; and is optionally substituted by at        least one group selected from hydroxyl, amino, carboxyl, C₁₋₆        alkyl, aryl and C(O)R;    -   Y is H or a substituent defined by X;    -   Z is the position of attachment for the targeting portion of the        library;    -   R¹ through R⁴ are selected independently from H; carboxyl; C₁₋₄        alkyl; C₁₋₄ alkyl substituted with a group selected from        hydroxyl, amino, sulfhydryl, halogen, carboxyl, C₁₋₄        alkoxycarbonyl and aminocarbonyl; an alpha carbon side chain of        a D- or L-amino acid other than proline; and C(O)R;    -   T is carbonyl or CH₂; and    -   M is metal for use in diagnostic imaging or an oxide or nitride        thereof.

The most preferred chelator for ^(99m)technetium radiopharmaceuticals isRP455. RP414 may also be used. The structures of RP414 and RP455 are asfollows:

-   -   R=CH₂OH RP414    -   R=C(CH₃)₃ RP455

Re and Tc complexes of these chelators are isostructural. Also, thesechelators are advantageous because the chemistry of these compounds iswell understood and they form neutral Re and ^(99m)Tc complexes. It ispossible to label these chelators with Re or ^(99m)Tc in one easy step.In addition these chelators have the advantage of being applicable forconjugation to a variety of targeting molecules, being compatible withsolid phase synthesis.

Labeling of RP414 with ^(99m)Tc can be carried out either at ambient orelevated temperature, rapidly, and with quantities of chelatorapproaching stoichiometric amounts. The complex is stable to both acidicand basic conditions and remains unchanged in-vivo.

Other chelators may be used to carry out the invention. The invention isnot limited to the preferred chelators listed above.

The chelator moiety of the targeting molecules incorporate adiagnostically useful metal within their structure as a complex.Suitable metals include radionuclides such as technetium and rhenium invarious forms such as ^(99m)TcO₃ ⁺, ^(99m)TcO₂ ⁺, ReO₃ ⁺, ReO₂ ⁺.Incorporation of the metal into the structure of the molecule can beachieved by various methods common in the art of coordination chemistry.In the case where the metal is technetium-99m (^(99m)Tc) the followinggeneral procedure may be used to generate the technetium-containingmolecule. The chelator is dissolved in an aqueous alcohol such asethanol to form a solution. The solution is then degassed to removedoxygen and the thiol protecting group removed with a suitable reagent,for example sodium hydroxide, and then neutralized with acetic acid. Inthe labeling step sodium petechnetate, obtained from a molybdenumgenerator, is added to the alcoholic solution of the chelator, togetherwith an amount of reducing agent sufficient to reduce the technetium,and the resulting solution heated. The labeled conjugate may beseparated from the contaminants ^(99m)TcO₄ ⁻ and colloidal ^(99m)TcO₂chromatographically, for example with a C-18 sep-pak cartridge.

In an alternative method, labeling may be effected by a transchelationreaction. The technetium source is a solution of technetium complexedwith labile ligands facilitating ligand exchange with the selectedchelator. Suitable ligands for transchelation are known to one skilledin the art and may include tartrate, citrate, glucoheptonate, andheptagluconate.

In a further alternative method, labeling may be accomplished utilizinga “one-pot” procedure whereby the sulfur protecting group is removedduring the labeling process. The molecule having an acetomidomethylgroup attached to the sulfur is dissolved in aqueous ethanol. In thelabeling step sodium petechnetate, obtained from a molybdenum generator,is added to the alcoholic solution of the chelator, together with anamount of reducing agent sufficient to reduce the technetium, and theresulting solution heated. The labeled conjugate may be separated fromthe contaminants ^(99m)TcO₄ ⁻ and colloidal ^(99m)TcO₂chromatographically, for example with a C-18 sep-pak cartridge.

A different approach to the labeling of the chelators defined above isdescribed in U.S. Pat. No. 5,789,555 and is incorporated herein byreference. The chelator molecules are immobilized on a solid phasesupport through a linkage that is cleaved upon metal chelation. This isachieved when the chelator is coupled to a functional group of thesupport by one of the complexing atoms. Preferably a complexing sulfuratom is attached to the support which is functionalized with a sulfurprotecting group such as maleimide.

When labeled with a diagnostically useful metal, molecules of thepresent invention can be used to detect sites of inflammation orinfection by procedures established by one skilled in the art ofdiagnostic imaging. Thus a molecule containing a metal such as ^(99m)Tcmay be administered to a mammal by intravenous injection in apharmaceutically acceptable solution such as isotonic saline. The amountof metal-containing molecule appropriate is dependent upon thedistribution profile of the chosen molecule in the sense that a compoundthat is cleared rapidly may be administered in higher doses than amolecule that clears less rapidly. Unit doses acceptable for imaginginflammation or infection are in the range of about 540 mCi for a 70 kgindividual. In vivo distribution and localization is tracked by standardscintigraphic techniques at an appropriate time subsequent to theinjection—typically between 30 minutes and 180 minutes depending uponthe rate of accumulation at the target site with respect to the rate ofclearance at non-target tissue.

Capping groups within the scope of the present invention include but arenot limited to:

Wherein Cl represents the point of attachment of the capping group.

Attaching the capping group to the targeting compound is achieved by theformation of an amide bond between the terminal carbon molecule of thecapping group and the amino group of the amino acid of the targetingcompound to which the capping group is attached. The Cl dissociates fromthe terminal carbon of the capping group upon formation of the amidebond.

Preferred capping groups have an amide group or a carbamate group. Thecapping group has a functionality that makes the entire compound eitheran antagonist or only a weak agonist of chemotaxis.

To increase the rate of identification of molecules that are tightlybound to the formylpeptide receptor, but which antagonize the effect offMLF at that receptor, it is desirable to employ techniques establishedby one skilled in the art of combinatorial chemistry. In view of theuncertainty associated with the effect of incorporation of a metal intothe molecular structures, it is also desirable to incorporate the metalinto the molecule before biological testing. Hence mixtures (orlibraries) of compounds are produced that contain a metal and these aretested in biological assays using techniques established to one skilledin the art of pharmacology. The inclusion of the metal into the moleculeat an early stage avoids uncertainty associated with testing unlabeledmolecules in biological assays in order to identify leads, and thenincorporating the metal. Hence the molecules tested in the mixture arestructurally identical to the final labeled molecule, but arenon-radioactive, thereby allowing easier testing. Promising mixtures areidentified using these assays and the constituent molecules prepared andre-tested.

Following initial selection of a suitable target molecule, a moderatelysized focused library of non-radioactive rhenium compounds is preparedas mixtures of up to 25 compounds. Typically, a large library of rheniumtargeting moiety conjugates is delivered as equimolar mixtures of 9-25compounds in 96 well microtiter plates (1 mg/well) for in vitro testing.These are then tested in the relevant assays and the most promisingmixtures are segregated for deconvolution. Depending on the number ofpromising molecules, discovered, a second round of testing may then beundertaken using a smaller subset of the rhenium containing moleculestogether with a second set of biological tests to further reduce thenumber of molecules. The final iteration will provide a series ofdiscrete compounds as both the rhenium complex and a free chelator readyfor labeling with radioactive ^(99m)technetium which is isostructural tothe non-radioactive rhenium isotope used. The potential imaging leadcandidates (preferably about 10 compounds) are delivered as purechelator targeting moiety conjugates for radiolabeling development andin vivo studies. This process provides labeled compounds that areeffective for binding a biological target in a rapid and cost effectivemanner.

The preferred targeting compounds of the present invention correspond toformula II. The preferred targeting compounds have the following formulaIII when a metal is attached to the chelator:

wherein CG is a capping group that makes the compound an antagonist or aweak agonist to chemotaxis; X, X2, X3 and X4 are amino acids selectedfrom natural and unnatural amino acids; X3 being a site of attachmentfor said chelator; R, and R2 is a linear or branched, saturated orunsaturated C₁₋₆ alkyl chain that is optionally interrupted by one ortwo heteroatoms selected from N, O, and S; and is optionally substitutedby at least one group selected from hydroxyl, amino, carboxyl, C₁₋₆alkyl, aryl and C(O)R; R3 is selected from H; alkyl; an alkylsubstituted by a group selected from amino, aminoacyl, carboxyl,guaniginyl, hydroxyl, thiol, phenyl, phenolyl, indolyl, and imidazolyl;M is a metal.

The most preferred targeting compound has the following formula IV:

-   -   Wherein M is 99mTc or Re

Methods and Materials

Peptide Synthesis

The effect of N-terminal capping groups on chemotactic peptide functionand binding characteristics was investigated by the preparation of thepeptide norleucyl-leucyl-phenylalanyl-lysine-COOH with the attachment ofseveral capping groups to the N-terminus as described above.

The various peptide sequences containing varying side chain protectinggroups in these examples were synthesized via a solid phase synthesismethod on an automated synthesizer using FastMoc chemistry on 1.0 mmolscale. The C-terminus of the peptide was attached to the solid phase viathe sasrin linker. Prior to the addition of each amino acid residue (ormixture of acids as described above) to the N-terminus of the peptidechain the FMOC group was removed with 20% piperidine in NMP. Each FMOCamino acid was activated with 0.5M HOBT/HBTU/DMF in the presence of 2.0MDIEA/NMP. After completion of the synthesis the resin was washed withNMP followed by dichloromethane and dried under vacuum for up to 24hours. Where mixtures of amino acids were employed the three amino acidswere added as equimolar mixtures of suitably side chain protected FMOCacid residues in a single coupling step and otherwise treated as asingle amino acid residue. Amino capping groups were added as describedin example 1.

Synthesis ofReO-Dimethylglycine-t-butyl-glycine-S-Acetamidomethyl-Cysteine-Glycine(ReO-RP455)Tetrafluorophenyl Ester

To ReO-RP455 (60 mg) in 1:1 acetonitrile:water (1 mL) was addedtetrafluorophenol (100 mg). The solution was diluted with acetonitrile(2 mL). The pH was adjusted to 2. To the solution was added1(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride. The reactionwas swirled to dissolve and the pH adjusted to 5. The reaction wasallowed sit at room temperature for 15 minutes followed by concentratingto a dark oil in vacuo. The product was purified on a Supelco supercleanLC-18 column. The column was first washed with a 5% acetonitrile: 95%water solution acidified to pH2 with 3N HCl. The product was eluted in a50% acetonitrile: 50% water solution acidified to pH2 with 3N HCl. Theappropriate pure fractions were identified by silica TLC(t-butanol:water:methanol, 10:3:2, rf: 0.85) followed by KMnO₄ staining.The correct fractions were pooled and concentrated in vacuo to ared-brown glass (58 mg).

Synthesis of Libraries of Rhenium-Containing Molecules on Sasrin Resin

Each of the N-terminus capped libraries on sasrin resin (20 mg) wasplaced in a Biorad disposable column. The Dde epsilon amino groupprotection on C-terminus Lysine was first removed with three washes of2% hydrazine in N-methylpyrrolidone (3×1 mL). The resin was thoroughlywashed with N-methylpyrrolidone then dichloromethane, and dried invacuo. To each vessel was addedReO-Dimethylglycine-t-butyl-glycine-S-Acetamidomethyl-Cysteine-Glycine(ReO-RP455)tetrafluorophenyl ester (10 mg) in ethyl acetate (1 mL). The reactionswere capped and shaken 20 hours at room temperature, followed byfiltration, washing with copious ethyl acetate, N-methylpyrrolidone,dichloromethane. The red-brown resins were dried in vacuo.

Cleavage of Rhenium-Containing Molecule Mixtures

Each of the ReO libraries were liberated from the supports in 95% TFA:5% water (1 mL) after 4 hours shaking at room temperature, followed byfiltration. The products were concentrated in vacuo. The residue wasredissolved in trifluoroacetic acid (150 uL) and dripped intot-butyl-methyl ether (5 mL) to precipitate. Each was centrifuged to apellet, the solvent decanted and the pellets dried in vacuo. Theproducts were dissolved in water and acetontrile (˜5 mL) and lyophilizedto pale pink powders.

Preparation of Single Molecules Containing Rhenium

Analysis of the results of the biological testing of the mixtures ofrhenium-containing molecules identified some mixtures as having higherbinding affinity than others have. The mixture having the highestaffinity was chosen for further deconvolution. The molecules wereprepared according to the methods used to prepare the mixtures ofmolecules, with the mixed amino acids being replaced by single aminoacids. The rhenium-containing molecules, once cleaved from the resin,were purified by reverse phase HPLC to give pure molecules exhibitingthe following physical properties:

-   ReO-CyclopropylCONH-nleu-leu-phe-trp-glu-lys(dimethylgly-t-butylgly-cys-gly)-gly-OH    HPLC retention time: 22 min; ESMS (1518, M+H), expected 1518-   ReO-CyclopropylCONH-nleu-leu-phe-ser-glu-lys(dimethylgly-t-butylgly-cys-gly)-gly-OH    HPLC retention time: 19.8 min; ESMS (1420, M+H), expected 1420-   ReO-CyclopropylCONH-nleu-leu-phe-tyr-glu-lys(dimethylgly-t-butylgly-cys-gly)-gly-OH    HPLC retention time: 20.1 min-   ReO-CyclopropylCONH-nleu-leu-phe-trp-his-lys(dimethylgly-t-butylgly-cys-gly)-gly-OH    Gly-lys(Dimethylgly-t-Butylgly-cys-gly)-his-trp-phe-leu-nleu-NHCOcyclopropyl    HPLC retention time: 21.0 min; ESMS (1528, M+H), expected 1528-   ReO-CyclopropylCONH-nleu-leu-phe-ser-his-lys(dimethylgly-t-butylgly-cys-gly)-gly-OH    HPLC retention time: 19.2 min; ESMS (1515, M+H), expected 1515-   ReO-CyclopropylCONH-nleu-leu-phe-tyr-his-lys(dimethylgly-t-butylgly-cys-gly)-gly-OH    HPLC retention time: 19.3 min; ESMS (1502, M+H), expected 1502-   ReO-CyclopropylCONH-nleu-leu-phe-trp-lys-lys(dimethylgly-t-butylgly-cys-gly)-gly-OH    HPLC retention time: 20.5 min; ESMS (1517, M+H), expected 1517-   ReO-CyclopropylCONH-nleu-leu-phe-ser-lys-lys(dimethylgly-t-butylgly-cys-gly)-gly-OH    HPLC retention time: 18.8 min; ESMS (1417, M+H), expected 1417-   ReO-CyclopropylCONH-nleu-leu-phe-tyr-lys-lys(dimethylgly-t-butylgly-cys-gly)-gly-OH    ESMS (1495, M+H), expected 1495    Animals and Reagents.

Sprague-Dawley rats weighing 300-350 g were purchased from CharlesRiver-Bausch & Lomb Laboratories (St. Constant, Quebec). Procedures werein accordance with standard Animal Care Committee protocols. Thefollowing compounds were used in this study: fMLF,N-tert-butyloxycarbonylated-methionyl-leucyl-phenylalanine (N-t-BocMLF),cytochalasin B, oyster shell glycogen, polyethylenimine,o-phenylenediamine (OPD), H₂O₂, H₂SO₄, sodium chloride tablets (SigmaChemical Corp., St. Louis, Mo.) and ³H-fMLF (New England Nuclear,Boston, Mass.). Peptide fMLP analogues,N-formyl-methionyl-leucyl-phenylalanyl-lysine (fMLFK), iso-BocMLFK,N-terminus capping groups 2-4, 6, 7, 10, 12, 13-21 attached to aminoacid sequence norleucyl-leucyl-phenylalanyl-lysine (NleLFK); librariesReORP552 to 557, deconvolution compounds ReORP553 17-01 to 17-09 andRP553 17-01 were synthesized in-house by Resolution Pharmaceuticals Inc.chemists (Mississauga, ON). fMLF peptide, N-t-BocMLF and fMLF peptideanalogues were stored at −20° C. in powder form and were diluted priorto each experiment in 2:1 acetonitrile:H₂O. N-terminal capping groupsattached to peptide backbone NleLFK were dissolved in DMSO and stored at−20° C. prior to use.

Rat Neutrophil Elicitation and Isolation.

Animals were sacrificed via administration of CO₂ 4 hours followingperitoneal injection of 10 mL of 0.5% (w/v) oyster glycogen warmed toroom temperature. Leukocyte harvesting via peritoneal lavage wasperformed using 20 mL of Hanks' buffered salt solution (HBSS−)containing 10 mM ethylene-diaminetetra-acetic acid (EDTA) disodium saltfollowed by a second lavage with 10 mL of HBSS− containing 10 mM EDTA.Exactly the same technique was performed on all rats with washes of theperitoneum prior to aspiration of samples approximating 1 minute. Thevolume of fluid recovered from each rat was approximately 20-25 mL andbloody lavages visibly containing significant red blood cell (RBC)populations were discarded.

Neutrophils isolated by peritoneal lavage were washed twice with 1 mL ofHBSS− (without calcium chloride, magnesium chloride and magnesiumsulfate) and centrifuged for ten minutes at 2000 rpms following eachwash. PMNs utilized in functional assays were centrifuged at 25° C.while those used in binding assays were centrifuged at 4° C. to preventinternalization. A cold H₂O RBC lysis was then performed with theaddition of 9 mL of sterile ice-cold H₂O and 1 mL of phosphate bufferedsaline (PBS) solution containing 0.1 M phosphate buffer, 0.027 M KCl and1.37 M NaCl following resuspension of the sample in 1 mL of HBSS.Centrifugation was subsequently performed for ten minutes at 1600 rpms.White blood cell (WBC) differential stain containing saline, 2% acidicacid and phenol violet dye was used to identify the neutrophilpopulation and trypan blue exclusion was used to determine viability ofcells. As previously reported, 98% of leukocytes were neutrophils andapproximately 95% of the cells were viable (Chen et al., 1996).

Human Neutrophil Isolation from Whole Blood.

Approximately 30 mL of venous blood was collected from healthyvolunteers with 100 units of heparin per mL of blood. Samples were thenmixed with 8 mL of 6% dextran solution and incubated at 37° C. for 1hour for RBC sedimentation. Leukocyte-rich plasma was collected andcentrifuged at 1500 rpm for 5 minutes. Cell pellets were resuspended in35 mL of saline and 10 mL of lymphocyte separation medium was layeredover prior to additional centrifugation at 1200 rpm for 30 minutes.Sedimented cells were washed once with 35 mL of saline and RBCs werelysed by resuspension in 1.8 mL of sterile H₂O for 15 seconds.Isotonicity was restored with the addition of 0.2 mL of 10×HBSS−followed by the addition of 35 mL of saline. Centrifugation at 1500 rpmfor 5 minutes was then performed. Following final washing of the PMNpellet with 35 mL of saline, cell viability was determined via trypanblue exclusion and cell number was determined.

Neutrophil fMLP Receptor Binding Assays.

K_(D) values were determined with fMLP saturation binding experimentsusing 2.5×10⁵ PMNs per well suspended in a final Volume of 150 uL offMLP, ³H-FMLP and/or HBSS+ in polypropylene 96-well plates. Eachcondition was performed in quadruplicate and non-specific binding wasassessed in the presence of PMNs, 10 uM FMLP and ³H-FMLP in the range of1 nM to 150 nM. Total binding was evaluated following the addition of³H-fMLP in the concentration range of 1 nM to 150 nM for development ofa saturation curve. The total amount of activity added to each samplewas determined from counts per minute (cpms) obtained from a samplecontaining 50 nM H³-fMLF. In all binding assays, specific binding wasexpressed in fmol and defined as difference between total binding andnon-specific binding.

Competition binding assays for N-terminal capping groups andRe-libraries were conducted with 6 nM ³H-fMLP in addition to theunlabelled competing analogue added at 1.0 uM and 10 uM to each well.For IC₅₀ value determination, the concentration of library mixtureReORP553 17 deconvolution compounds, ranged from 10⁻¹²M to 10⁻³M in thepresence of 6 nM ³H-fMLF. Total binding in the competition assays wasassessed in the presence of 1.0×10⁶ PMNs per sample while non-specificbinding was determined in the absence of cells. Each condition wasperformed in quadruplicate. Total activity added to each sample wasdetermined from cpms obtained from a sample containing 6 nM ³H-fMLF.Data was expressed as specific binding in fmols and converted to percentof total ³H-fMLF binding (% control).

After a 1 hour incubation period at 4° C., the samples were harvestedwith the Tomtec Mach III Cell Harvester by vacuum aspiration onto 1.5 umpore size Skatron glass-fibre filter mats pre-treated with 0.1%polyethylenimine (w/v) for approximately 24 hours. Sample wells receivedthree consecutive 12 second washes with 0.9% saline solution and 5 mL ofliquid scintillation fluid was added to collected filters. Filters werecounted for 2 minutes in the Beckman β-counter following exposure toscintillation fluid for 15 hours. Data was expressed in terms ofspecific binding in fmols converted to percent of total ³H-fMLF binding(% control).

Measurement of Myeloperoxidase Release.

0.5×10⁶ PMNs per sample were incubated in 96-well Millipore Multiscreen0.65 um filter plates. In a final volume of 150 uL, 50 uL of therespective fMLP analogues (10 uM to 1 pM) and/or fMLP (10 uM to 1.0 uM)were incubated with isolated PMNs pre-treated with cytochalasin B (5ug/mL) for 10 minutes. Following a 30 minute incubation period at roomtemperature, the supernatant was collected into a 96-well polypropyleneplate with the Millipore vacuum apparatus. A stock solution ofo-phenylenediamine (OPD) was made to contain 2.5 mg of OPD, 5 ul of H₂O₂and 10 mL HBSS+ and supernatant samples were subsequently incubated with50 uL of OPD solution. The reaction was stopped after 2 minutes by theaddition of 2.5M H₂SO₄. Photometric analysis was performed with theThermomax Microplate Reader with optical density (O.D.₄₉₀) values beingread at a wavelength of 490 nm. Absorbance was measured against a blankcontaining 150 μl of HBSS+ which was subtracted from wells containingcells and the various treatments. MPO release was expressed as apercentage of the total release of myeloperoxidase in control wellscontaining cells ±fMLF.

Radiolabeling of RP553 17-01

RP553 17-01 (200 ug; 0.144 mmoles) was dissolved in 100 ul saline and100 ul acetonitrile. Tin (II) chloride (40 ug) and sodium gluconate (1.3mg) was added to the peptide solution followed by Na[⁹⁹TcO4] (10 mCi).The reaction was carried out for one hour at room temperature. Thereaction was analyzed using high performance liquid chromatography(HPLC) and the product was purified under a step-gradient condition.

Data Analysis

The curve-fitting program Prism was used to calculate K_(D), IC₅₀ andEC₅₀ values by fitting data to the one-site binding equations A, B and Crespectively.

-   A. Equation: One site binding (hyperbola)    Y=Bmax*X/(K _(D) +X)-   B. Equation: One site competition    Y=Bottom+(Top−Bottom)/(1+10^((X−LogIC50))); with x=log    (concentration) and y=binding-   C. Equation: Sigmoidal dose-response (variable slope)    Y=Bottom+(Top−Bottom)/(1+10^(((LogEC50−X)*Hill Slope))))    The goodness of fit for data to the above equations was determined    by the correlation coefficient (with r=1 being a perfect fit) and    95% confidence intervals were calculated for IC₅₀ and EC₅₀ values.    Additionally, Graph fit software was used to perform Scatchard    analysis of saturation binding data and to confirm K_(D) and Bmax    values generated by Prism software.

Individual experiments (denoted by n values) were performed inquadruplicate to account for intra-assay variability. Results areexpressed as means±S.E.M. unless otherwise indicated. Statisticalsignificance among multiple groups was evaluated with a one way analysisof variance while a student's t-test was used to evaluate significancebetween sample treatments. A p value of <0.05 was consideredsignificant.

Experimental Results

Library Design

Based on capping group data, a combination of parallel synthesis andsplit and mix technologies was used to prepare a combinatorial libraryof 324 Re-peptides. Specifically the library was composed of 36 mixturescontaining equimolar quantities of 9 different compounds for analysis inbinding studies based on the following peptide sequence:

Where A, B, C, D, E, F are natural amino acids and the capping groups(CG) are selected from those above. The following table indicates theamino acids and capping groups used in individual libraries. TABLE 1Amino acid substitutions and N-terminal capping groups for Re-LibraryMixtures Mixture Mixture RP# A, B, C D, E, F Capping Group 552-6 Phe,Asp, Leu Trp, Ser, Tyr N,N-diethycarbamyl 552-10 Phe, Asp, Leu Trp, Ser,Tyr N-phenyl,N-methylcarbamyl 552-13 Phe, Asp, Leu Trp, Ser, TyrAdamantylcarbonyl 552-16 Phe, Asp, Leu Trp, Ser, TyrFluorenylmethylcarbonyl 552-17 Phe, Asp, Leu Trp, Ser, TyrCyclopropylcarbonyl 552-18 Phe, Asp, Leu Trp, Ser, TyrN,N-diphenylcarbamyl 553-6 Glu, His, Lys Trp, Ser, TyrN,N-diethycarbamyl 553-10 Glu, His, Lys Trp, Ser, TyrN-phenyl,N-methylcarbamyl 553-13 Glu, His, Lys Trp, Ser, TyrAdamantylcarbonyl 553-16 Glu, His, Lys Trp, Ser, TyrFluorenylmethylcarbonyl 553-17 Glu, His, Lys Trp, Ser, TyrCyclopropylcarbonyl 553-18 Glu, His, Lys Trp, Ser, TyrN,N-diphenylcarbamyl 554-6 Asn, Arg, Val Glu, His, LysN,N-diethycarbamyl 554-10 Asn, Arg, Val Glu, His, LysN-phenyl,N-methylcarbamyl 554-13 Asn, Arg, Val Glu, His, LysAdamantylcarbonyl 554-16 Asn, Arg, Val Glu, His, LysFluorenylmethylcarbonyl 554-17 Asn, Arg, Val Glu, His, LysCyclopropylcarbonyl 554-18 Asn, Arg, Val Glu, His, LysN,N-diphenylcarbamyl 555-6 Phe, Asp, Leu Asn, Arg, ValN,N-diethycarbamyl 555-10 Phe, Asp, Leu Asn, Arg, ValN-phenyl,N-methylcarbamyl 555-13 Phe, Asp, Leu Asn, Arg, ValAdamantylcarbonyl 555-16 Phe, Asp, Leu Asn, Arg, ValFluorenylmethylcarbonyl 555-17 Phe, Asp, Leu Asn, Arg, ValCyclopropylcarbonyl 555-18 Phe, Asp, Leu Asn, Arg, ValN,N-diphenylcarbamyl 556-6 Trp, Ser, Tyr Asn, Arg, ValN,N-diethycarbamyl 556-10 Trp, Ser, Tyr Asn, Arg, ValN-phenyl,N-methylcarbamyl 556-13 Trp, Ser, Tyr Asn, Arg, ValAdamantylcarbonyl 556-16 Trp, Ser, Tyr Asn, Arg, ValFluorenylmethylcarbonyl 556-17 Trp, Ser, Tyr Asn, Arg, ValCyclopropylcarbonyl 556-18 Trp, Ser, Tyr Asn, Arg, ValN,N-diphenylcarbamyl 557-6 Ile, Gln, Thr Asn, Arg, ValN,N-diethycarbamyl 557-10 Ile, Gln, Thr Asn, Arg, ValN-phenyl,N-methylcarbamyl 557-13 Ile, Gln, Thr Asn, Arg, ValAdamantylcarbonyl 557-16 Ile, Gln, Thr Asn, Arg, ValFluorenylmethylcarbonyl 557-17 Ile, Gln, Thr Asn, Arg, ValCyclopropylcarbonyl 557-18 Ile, Gln, Thr Asn, Arg, ValN,N-diphenylcarbamylCompetition Binding of Rhenium-Libraries

Potential capping group modifications identified as antagonists(6,10,13,16,17) or agonists with reduced activity (18) in functionalscreens as well as in preliminary binding assays were implemented intothe library design. The application of combinatorial chemistry allowedfor high-throughput screening of a library of 36 mixtures consisting ofequimolar concentrations of 9 compounds all of which were complexed toRe. Each library (labeled 552 to 557) varied in the specific amino acidsincorporated at position 4 and 5 from the N-terminus. Furthermore,additional variation within a single library (e.g. 552) was introducedwith the addition of varied N-terminal capping groups.

Competition binding data of libraries at 1.0 uM and 10 uM with 6 nM³H-fMLF indicate dose-dependent inhibition of ³H-fMLF binding in allcases as shown in FIGS. 1A and 1B respectively). It should be noted thatthe control compound (designated 455 on FIGS. 1A and 1B) representsbinding by Re-metal complexed to the chelator(Dimethylgly-t-Butylgly-cys-gly-). This negative control did not inhibit³H-fMLF binding to any significant degree. Several mixtures(ReORP552-17, ReORP553-17, ReORP555-17, ReORP556-17, ReORP555-18, etc.)exhibit significant displacement of ³H-fMLF whereas other mixturesexhibit relatively weak binding properties (ReORP554-17, ReORP554-10,ReORP557-13, etc.) This variation was used to decide which mixturesrequire deconvolution into 9 separate compounds for further testing.

Deconvolution of Mixture ReORP553 17

Library mixture ReORP553 17 showing the highest binding affinity (lowestfMLP remaining) in competition binding assays (RP553-17) was selectedand the constituent single peptides were prepared and isolated as purecompounds. The following is a list of the corresponding amino acidsequences for individual Re-peptides:

RP553-17-01

-   ReO-CyclopropylCONH-nleu-leu-phe-trp-glu-lys(dimethylgly-t-butylgly-cys-gly)-gly-OH    HPLC retention time: 22 min; ESMS (1518, M+H), expected 1518    RP553-17-02-   ReO-CyclopropylCONH-nleu-leu-phe-ser-glu-lys(dimethylgly-t-butylgly-cys-gly)-gly-OH    HPLC retention time: 19.8 min; ESMS (1420, M+H), expected 1420    RP553-17-03-   ReO-CycopropylCONH-nleu-leu-phe-tyr-glu-lys(dimethylgly-t-butylgly-cys-gly)-gly-OH    HPLC retention time: 20.1 min    RP553-17-04-   ReO-CyclopropylCONH-nleu-leu-phe-trp-his-lys(dimethylgly-t-butylgly-cys-gly)-gly-OH    HPLC retention time: 21.0 min; ESMS (1528, M+H), expected 1528    RP553-17-05-   ReO-CyclopropylCONH-nleu-leu-phe-ser-his-lys(dimethylgly-t-butylgly-cys-gly)-gly-OH    HPLC retention time: 19.2 min; ESMS (1515, M+H), expected 1515    RP553-17-06-   ReO-CyclopropylCONH-nleu-leu-phe-tyr-his-lys(dimethylgly-t-butylgly-cys-gly)-gly-OH    HPLC retention time: 19.3 min; ESMS (1502, M+H), expected 1502    RP553-17-07-   ReO-CyclopropylCONH-nleu-leu-phe-trp-lys-lys(dimethylgly-t-butylgly-cys-gly)-gly-OH    HPLC retention time: 20.5 min; ESMS (1517, M+H), expected 1517    RP553-17-08-   ReO-CyclopropylCONH-nleu-leu-phe-ser-lys-lys(dimethylgly-t-butylgly-cys-gly)-gly-OH    HPLC retention time: 18.8 min; ESMS (1417, M+H), expected 1417    RP553-17-09-   ReO-CyclopropylCONH-nleu-leu-phe-tyr-lys-lys(dimethylgly-t-butylgly-cys-gly)-gly-OH    ESMS (1495, M+H), expected 1495    Competition Binding of Mixture ReORP553 Deconvolution Compounds

Although several Re-libraries exhibited significant displacement of 6 nM³H-fMLF, library ReORP553 17-01 was chosen for deconvolution due tohigh-degree of ³H-fMLF displacement exhibited at 10 uM. Deconvolutioncompound synthesis yielded eight ReO-peptides exhibiting purity greaterthan 80%. Complexation of rhenium to the final ninth peptide (ReORP55317-03) of this mixture could not be successfully synthesized with due torelatively low yield so it is not indicated in the deconvolutionresults.

All nine N-terminal cyclopropanecarbonyl-modified peptides with varyingamino acid sequences exhibited dose-dependent competition in thereceptor binding assays. For comparative measures, fMLF and N-t-BocMLFwere evaluated as representative agonist and antagonist standardsrespectively. Competition binding curves for deconvolution compounds areexhibited in FIGS. 1A-H and respective IC₅₀ values derived from thesecurves are summarized in Table V. Although none of the Re-peptidesexhibited greater binding affinity than fMLF, all of them were at least1.5-fold more potent than N-t-BocMLF. Furthermore, ReORP553 17-01exhibited the highest binding affinity which was approximately 12-foldgreater than known antagonist N-t-BocMLF but 15-fold less than fMLF. Thestructure and corresponding amino acid sequence of ReORP553 17-01 is asfollows:

TABLE 2 Rat FPR Binding for deconvolution compounds of library ReORP55317 Deconvolution Receptor Binding Compound (IC₅₀, uM) n value fMLF 0.05(0.008 to 0.27) 3 N-t-BocMLF  9.3 (4.21 to 20.57) 4 ReORP553 0.73 (0.60to 0.88) 3 17-01 ReORP553 2.31 (1.41 to 3.79) 3 17-02 ReORP553 1.77(1.30 to 2.42) 3 17-04 ReORP553 1.01 (0.53 to 1.93) 3 17-05 ReORP5531.22 (0.62 to 2.4) 2 17-06 ReORP553 0.76 (0.19 to 3.0) 2 17-07 ReORP5534.95 (1.78 to 13.71)  2* 17-08 ReORP553 5.93 (2.96 to 11.91)  1* 17-09Binding of ReORP553 17-01 to Human Neutrophils

Competition binding curves of ReORP553 17-01, fMLF and N-t-BocMLF tohuman neutrophils are shown in FIG. 3. Respective IC₅₀ values are shownin Table 3. While fMLF exhibits the lowest IC₅₀ value, values forReORP553 17-01 and N-t-Boc indicate similar binding affinities. TABLE 3Human FPR Binding of ReORP553 17-01 Compound IC₅₀ Value (uM) fMLF 0.005(0.0009 to 0.03) ReORP553 17-  0.14 (0.008 to 2.4) 01 N-t-BocMLF  0.18(0.03 to 1.04)Data is expressed as IC₅₀ values (with 95% confidence intervals) (n = 1,performed in triplicate) See FIG. 7 for associated receptor bindingcurves.Effect of ReORP553 17-01 on Myeloperoxidase Release from HumanNeutrophils

ReORP553 17-01 significantly inhibited the release of MPO in humanneutrophils stimulated with fMLF at concentrations of 10⁻⁸ M and 10⁻⁶ M(p<0.05) (FIG. 4). Although not shown on FIG. 4, data pertaining toadministration of 10⁻¹⁴M fMLF coincided with data for 10⁻¹²M fMLF inthat there was no significant increase in MPO release for any of thetreatments. Specifically, ReORP553 17-01 administered at a concentrationas high as 10 uM did not stimulate MPO release that indicates that itmay be an antagonist. Known antagonist N-t-BocMLF was used as aninternal standard and correspondingly inhibited MPO release from fMLFstimulated cells and did not induce release at lower concentrations offMLF.

Radiolabelling of RP553 17-01 with Technetium-99m

RP553 17-01 was successfully radiolabeled to yield ^(99m)Tc-RP553 17-01with >90% radiochemical purity (see FIG. 5).

Saturation and Scatchard Analysis of Neutrophil fMLF Receptor Binding

Specific binding of [³H]-fMLF to peritoneal rat neutrophils wascharacterized with total [³H]-fMLF concentrations ranging from 1 to 125nM in the presence and absence of an excess of cold fMLF (FIG. 6A).Analysis of the saturation binding data by Prism software indicated aK_(D) value of 6.1±2.3 nM and 4.0±0.9×10⁴ binding sites expressed percell (n=3) (Table 4). Scatchard analysis (FIG. 6B) suggested thepresence of a single class of binding sites (r=−0.97) and confirmedanalysis with Prism software with a corresponding K_(D) value of6.4±0.02 nM and 4.1±0.06×10⁴ binding sites per cell. TABLE 4 fMLFreceptor binding on rat neutrophils Binding Sites/Cell K_(D) (nM) nControl 4.0 ± 0.9 × 10⁴ 6.1 ± 2.3 3B_(max) values used to estimate binding sites per cell and the K_(D)values were derived from one-site binding equation in Prism software.Scatchard analysis confirmed these results with a K_(D) of 6.4 ± 0.02 nMand a 4.1 ± 0.06 binding sites per cell. (values expressed as mean ±S.E.M.; n = 3 performed in quadruplicate).Dose-Response of Myeloperoxidase Release by fMLF and fMLFK

Photometric measurement of myeloperoxidase release from elicited ratPMNs in response to known agonists fMLF and fMLFK indicated adose-dependent response (FIGS. 7A and 7B respectively). EC₅₀ values(with 95% confidence intervals) of 0.11 (0.04 to 0.28) uM and 0.14 (0.13to 0.16) uM for fMLF and fMLFK respectively did not indicate significantdifferences in agonist potency (p>0.05).

3.3 Effect of fMLF Amine Capping Groups on Myeloperoxidase Release

Several N-terminal capping groups attached to the amino acid backboneNleLFK, were assessed in functional assays to determine agonist orantagonist activity in terms of MPO release. Dose-dependent increases inMPO release were observed with peptides modified with3-but-1-enoxycarbonyl, 2-acetamido-5-thiazolesulfonyl, 2-quinoxazoyl andtrans-cinnamoyl groups indicating agonist activity (Table 5). AlthoughN-terminal modification of the peptide backbone withN,N-diphenylcarbamoyl did not cause a dose-dependent increase in MPOrelease, the resultant stimulation at the 10 uM concentration was stillsignificant (p<0.05). It should be noted that these compounds were notas potent in stimulating MPO release as the agonist standards fMLF andfMLFK at 1 uM and 10 uM concentrations (See FIG. 7). TABLE 5 Chemicalidentification and functional data of amine capping groups displayingagonist function in relation to control values Capping % Control %Control Group # Chemical Identification (1 uM) n P (10 uM) N p 73-but-1-enoxycarbonyl 133.8 ± 13.3 2 <0.05 229.3 ± 13.3 2 <0.05 122-acetamido-5- 117.5 ± 7.8  2 >0.05 175.5 ± 12.0 2 <0.05thiazolesulfonyl 14 2-quinoxazoyl 117.2 ± 18.6 2 >0.05 129.8 ± 6.0  2<0.05 15 trans-cinnamoyl 133.1 ± 10.3 2 <0.05 195.8 ± 14.3 2 <0.05 18N,N-diphenylcarbamoyl 142.7 ± 8.8  2 <0.05 125.2 ± 1.5  2 >0.05Control wells consisted of PMNs without any additional ligand presentwhile amine capping groups were administered at 1 uM and 10 uMconcentrations. Data is expressed as % control ± S.E.M. and n valuesrepresent samples performed in quadruplicate.

Table 6 displays functional data pertaining to amine capping groupmodifications which did not significantly increase the degree of MPOrelease in relation to unstimulated PMNs. Specifically, the lack of PMNstimulation exhibited by 2-methoxyacetyl (2), furoyl (3),phenylthioacetyl (4), N,N-diethylcarbamoyl (6) and cyclopropylcarbonyl(17)-NleLFK peptides suggest these modifications result in non-bindingor possibly anatgonist ligands. However, competition binding assays(FIG. 8) with N,N-diethylcarbamoyl-NleLFK and cycloproylcarbonyl-NleLFKindicate that these ligands inhibit ³H-fMLF binding and are thereforemore likely antagonists. Antagonist activity was also observed withN-phenyl-N-methylcarbamoyl(10), adamantanecarbonyl (13) andfluorenylmethylcarbonyl(16) capping groups which significantly decreasedbasal levels of MPO release resulting from the PMN elicitation process.It should be noted that the degree of MPO release in unstimulated cellswas not significantly different from PMNs exposed to known antagonistsisoBOC-MLFK and N-t-BocMLFK at concentrations of 1.0 uM and 10 uM. TABLE6 Chemical identification and functional data of fMLF amine cappinggroups which displayed non-binding or antagonist characteristicsrelative to control values Capping % Control % Control Group # ChemicalIdentification (1 uM) N P (10 uM) n p 2 2-methoxyacetyl 95.0 ± 15.72 >0.05 97.1 ± 37.1 2 >0.05 3 Furoyl 93.0 ± 9.0  3 >0.05 102.5 ± 9.0 3 >0.05 4 Phenylthioacetyl 138.7 ± 28.6  2 >0.05 119.7 ± 20.3  2 >0.05 6N,N-diethylcarbamoyl 113.8 ± 14.3  3 >0.05 109.6 ± 3.3  3 >0.05 10N-phenyl-N- 94.7 ± 20.4 2 >0.05 68.0 ± 17.4 2 <0.05 methylcarbamoyl 13Adamantylcarbonyl 70.5 ± 14.9 2 <0.05 71.7 ± 11.2 2 <0.05 16Fluorenylmethylcarbonyl 84.1 ± 27.6 2 >0.05 59.9 ± 19.0 2 <0.05 17Cyclopropylcarbonyl 108.0 ± 14.5  3 >0.05 91.8 ± 16.3 3 >0.05N-t-BocMLFK 89.8 ± 32.5 2 >0.05 86.7 ± 31.1 2 >0.05 iso-BocMLFK 99.6 ±16.5 2 >0.05 94.7 ± 20.2 2 >0.05Control wells consisted of PMNs without any additional ligand while testcompounds were administered at 1 uM and 10 uM concentrations. Data isexpressed as % control ± S.E.M. and n values represent samples performedin quadruplicate.

In addition to decreasing basal levels of MPO release (Table 6), cappinggroups 10, 13, and 16 also exhibited antagonist function in fMLFstimulated cells (Table 7). Furthermore, capping group 6 inhibited MPOrelease at concentrations of 10 uM indicating that it is not anon-binding ligand but rather a potential antagonist. Although cappinggroup 14 significantly increased the degree of MPO release atconcentrations of 10 uM (Table 5), significant inhibition was exhibitedwhen administered at 10 uM in the presence of fMLF (p<0.05). Thisindicates possible partial agonist function. Known antagonistiso-Boc-MLFK exhibited similar functional characteristics as thesemodified peptides by significantly inhibiting fMLF-stimulated MPOrelease at concentrations of 10 uM. It should be noted that MPO releasedata for capping groups 2, 3, 4 and 17 was not included because internalstandard antagonist iso-BocMLFK did not exhibit typical inhibition ofMPO release as was seen in other assays. TABLE 7 Effect of fMLF aminecapping groups on myeloperoxidase release in elicited rat PMNsco-stimulated with 0.1 uM fMLF % Control % Control MPO release MPORelease % Control (1 uM capping (10 uM MPO release Capping group + 0.1uM capping group + 0.1 uM (0.1 uM fMLF) Group # Chemical IdentificationfMLF) fMLF) n 291.8 ± 33.0 6 N,N-diethylcarbamyl 283.3 ± 25.1 249.9 ±6.2  2 155.7 ± 28.5 10 N-phenyl,N- 171.2 ± 6.3  121.3 ± 23.9 2methylcarbamyl 155.7 ± 28.5 13 Adamantylcarbonyl 131.6 ± 30.9 128.6 ±40.9 2 324.6 ± 4.6  14 2-quinoxazoyl 323.2 ± 16.8 239.8 ± 9.0  2 155.7 ±28.5 16 Fluorenylmethylcarbonyl 161.7 ± 6.2  108.9 ± 28.5 2 324.6 ± 4.6 18 N,N- 321.6 ± 8.6  185.9 ± 15.7 1 diphenylcarbamoyl 212.0 ± 58.8iso-BocMLFK 215.4 ± 66.3 179.9 ± 50.6 3Control wells consisted of elicited rat PMNs without any additionalligand and fMLF was administered at 0.1 uM. In every case, fMLFsignificantly increased the release of myeloperoxidase in relation tocontrol samples in all cases (p < 0.05). A dose-dependent decrease inmyeloperoxidase release was observed in fMLF-stimulated PMNs uponadministration of all capping groups with significant inhibitionoccurring at 10 uM# (p < 0.05). Data is expressed as mean % control ± S.E.M. and n valuessamples performed in quadruplicate.3.4 Binding of fMLF Amine Capping Groups

As determined by saturation analysis 6 nM ³H-fMLF was used incompetition assays. Competition binding analysis of potentialantagonists and modest agonist compounds identified in functionalscreens indicate that peptide modifications of the N-terminus withcyclopropylcarbonyl (17) or N,N-diphenylcarbamyl (18) groups do notnullify binding affinity (FIG. 8). Moreover, at concentrations of 10 uM,these peptides inhibited ³H-fMLF binding to a degree comparable withknown antagonist iso-BocMLFK. In particular, this data should be notedfor capping group modification 17 since the available functional datawith PMNs lacking fMLF stimulation implies that it may be an antagonistor potentially a non-binding ligand. Therefore, additional binding datapertaining to the capping group modification 17 suggests that thispeptide may be an antagonist. Other peptides modifications such as withcapping groups 6, 10, 13 and 16 did not significantly decrease theamount of ³H-fMLF binding indicating relatively low binding affinity.However, these peptides did exhibit significant inhibition of MPOrelease at the 10 uM concentration in fMLF stimulated PMNs (Table 6).

Although the invention has been described with preferred embodiments, itis to be understood that modifications may be resorted to as will beapparent to those skilled in the art. Such modifications and variationsare to be considered within the purview and scope of the presentinvention.

REFERENCE LIST

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1-20. (canceled)
 21. A compound for binding to sites of inflammationhaving the following formula I:

wherein CG is a capping group selected from the group consisting of

wherein Cl represents the point of attachment of the capping group; X,X₂, X₃ and X₄ are amino acids selected from natural and unnatural aminoacids; Z is a chelator capable of complexing a radionuclide metal or achelator complexed to a radionuclide metal, X₃ being a site ofattachment for said chelator.
 22. A compound according to claim 21having the following formula II:

wherein R, and R₂ is a linear or branched, saturated or unsaturated C₁₋₆alkyl chain that is optionally interrupted by one or two heteroatomsselected from N, O, and S; and is optionally substituted by at least onegroup selected from hydroxyl, amino, carboxyl, C₁₋₆ alkyl, aryl andC(O)R; R₃ is selected from H; alkyl; an alkyl substituted by a groupselected from amino, aminoacyl, carboxyl, guaniginyl, hydroxyl, thiol,phenyl, phenolyl, indolyl, and imidazolyl.
 23. A compound according toclaim 21 wherein the compound binds toN-formyl-methionyl-leucyl-phenylalanine (fMLF) receptor.
 24. A compoundaccording to claim 21 wherein X₃ is Lys and X₄ is Gly.
 25. A compoundaccording to claim 24 having the following formula III:


26. A method for imaging sites of inflammation in a patient comprisingadministering a compound of claim 21 and imaging.
 27. The method ofclaim 26 wherein the compound has the following formula II:

wherein R, and R₂ is a linear or branched, saturated or unsaturated C₁₋₆aklyl chain that is optionally interrupted by one or two heteroatomsselected from N, O, and S; and is optionally substituted by t least onegroup selected from hydroxyl, amino, carboxyl, C₁₋₆ alkyl, aryl andC(O)R; R₃ is selected from H; alkyl; an alkyl substituted by a groupselected from amino, aminoacyl, carboxyl, guaniginyl, hydroxyl, thiol,phenyl, phenolyl, indolyl, and imidazolyl.
 28. The method of claim 27,wherein the compound has the following formula IV:

wherein M is ^(99m)Tc or Re.